The present invention relates to a previously unknown receptor protein which were isolated and identified based on specific expression of the T cell genes using a technique identified by the present inventor in a publication (Proc. Natl. Acad. Sci. USA. 84, 2896-2900, May 1987, Immunology), and more particularly relates to the receptor protein, 4-1BB, a monclonal antibody against 4-1BB, a ligand protein for detecting the presence of 4-1BB binding sites on cells and methods of using these proteins and antibody.
Lymphokines are the proteins by which the immune cells communicate with each other. Scientists produce them in sufficient quantities for therapeutic use against immunologic diseases. The immune system of humans and other species requires that white blood cells be made in the bone marrow, which white blood cells include phagocytes, lymphocytes and B cells. As presently understood, the phagocytes include macrophage cells which scavenge unwanted materials such as virus protein from the system. The lymphocytes include helper T cells and killer T cells and B cells as well as other cells, including those categorized as suppressor T cells.
The B cells produce the antibodies. The killer T cells physically pierce the cell and the helper T cells facilitate the whole process. In any event, the immune process is facilitated by lymphokines. Interleukin 1, secreted from macrophages activate the helper T cells and raise the body temperature causing fever which enhances the activity of the immune cells. The activated helper T Cells produce Interleukin 2 and Interleukin stimulates the helper and killer T cells to grow and divide. The helper T cells also produce another lymphokine, B cell growth factor (BCGF), which causes B cells to multiply. As the number of B cells increases, the helper T cells produce another lymphokine known as the B cell differentiating factor (BCDF), which instructs some of the B cells to stop replicating and start producing antibodies. T cells also produce a lymphokine, gamma interferon (IF), which has multiple effects like Interleukin 2. Interferon helps activate killer T cells, enabling them to attack the invading organisms. Like BCGF, interferon increases the ability of the B cells to produce antibodies. Interferon also affects the macrophages to keep them at the site of the infection and help the macrophages to digest the cells they have engulfed. Gathering momentum with each kind of lymphokine signal between the macrophages and the T cells, the lymphokines amplify the immune system response and the virus protein or other foreign matter on the infected cells is overwhelmed. There are many other lymphokines, maybe a hundred or more, which participate in the immune process. Many lymphokines are known and many are not.
Lymphokines are sometimes called intercellar peptide signals. Among scientists there is widespread use of cloned cell lines as lymphokine producers and the isolation of lymphokine mRNA has become a common technique. The present invention relates to a previously unknown receptor protein which was isolated and identified based on specific expression of the T cell genes using a technique identified by the present inventor in a publication (Proc. Natl. Acad. Sci. USA. 84, 2896-2900, May 1987, Immunology). The protocol reported in this publication can be used by scientists to detect virtually all of the lymphokines because the method is designed to detect virtually all the mRNA expressed differentially and the mRNA sequences of the immune cells are expressed differentially as they relate to the T cells and the killer T cells even though the level of expression is low and the quantity of the secreted lymphokine protein is low. The present inventor believes that the analysis described in the above identified publication can reveal biologically important molecules such as lymphokines because there are many indications that biologically important or active molecules are coded by the most scarce messages. An example is a transforming growth factor (TGF) which is present as only one of a million clones. There are many known lymphokine proteins and they include the interferons, interleukin-1,2,3,4,5,6,7, colony-stimulating factors, lymphotoxin, tumor necrosis factor and erythropoietin, as well as others.
Most T cell factors have been classically identified by recognizing biologic activities in assays, purifying the protein information. An alternative approach is to isolate putative T cell genes based upon specific expression and then demonstrate the function of the unknown molecule. Using the aforesaid modified differential screening procedure, the present inventor has recently cloned a series of T cell subset-specific cDNAs from cloned helper T (HTL) L2 and cloned cytolytic T lymphocyte (CTL) L3.
A series of T-cell subset-specific cDNAs were isolated from cloned murine T-cells by employing a modified differential screening procedure (88, 89). The nucleotide sequence and expression properties of some of the cDNA species have been reported (90). One of the genes not previously characterized, 4-1BB, was studied further. Apparent full length cDNAs corresponding to fourteen species of the 16 initial isolates were sequenced and were found to constitute five different species. Three of the five were identical to previously reported cDNA sequences of proenkephalin, T cell replacing factor and HF gene (a serine esterase). The other two, represented as L2G25B and 4-1BB, were novel sequences of unknown function. The open reading frames of 4-1BB and L2G25B code for 245 and 92 amino acids, respectively. The predicted proteins of 4-1BB and L2G25B include 22 and 23 amino acid-long putative signal sequences, respectively. The protein backbones of mature proteins encoded by 4-1BB and L2G25B are composed of 234 amino acids with molecular weight of 25000 and 69 amino acids with molecular weight of 7880, respectively. 4-1BB contains two potential N-glycosylation sites while L2G25B has none. 4-1BB contains 23 cysteine residues in the putative mature protein.
The cDNA L2G25B encodes for the lymphokine, macrophage inflammatory protein-1xcex1 or MIP-1xcex1. MIP-1xcex1 has been described in a paper entitled, xe2x80x9cEnhancing and Suppressing Effects of Recombinant Murine Macrophage Inflammatory Proteins on Colony Formation In Vitro by Bone Marrow Myeloid Progenitor Cellsxe2x80x9d, Hal E. Broxmeyer, Barbara Sherry, Li Lu, Scott Cooper, Kwi-Ok Oh, Patricia Tekamp-Olson, Byoung S. Kwon, and Anthony Cerami, Blood 76, 111-1116, 1990 and is incorporated herein by reference. This was the first time the suppressing activity of MIP-1xcex1 was characterized.
The cDNA clone, called 4-1BB, was originally believed to be a lymphokine based upon the early experiments disclosed herein. The later studies showed that 4-1BB is an inducible receptor-like sequence found in both cytolytic and helper T-cells. Chalupny and colleagues (132) published a paper disclosing a fusion protein consisting of the extracellular domain of 4-1BB and the Fc region of IgG1. Chalupny et al. taught that the highest levels of 4-1BB Rg (4-1BB-immunoglobulin fusion protein) binding was to human vitronectin. The present inventor performed an ELISA study using 4-1BB-AP (the fusion protein of the present invention) and human vitronectic. No binding of 4-1BB-AP based on alkaline phoshatase activity was observed. To rule out the possibility that 4-1BB-AP was binding to proteins extrinsically attached to the cell surface, B-Cell lymphomas were washed in acid conditions prior to the binding assay; 4-1BB-AP still bound specifically to mature B-cell lymphomas. Based on the data reported by Chalupny et al. it does not appear that they teach a fusion protein capable of accurately identifying 4-1BB ligands.
The present invention includes the receptor protein 4-1BB and the cDNA gene encoding for receptor protein 4-1BB. the nucleotide sequence of the isolated cDNA is disclosed herein along with the deduced amino acid sequence. The cDNA gene identified as p4-1BB was deposited at the American Type Culture Collection at 12301 Parklawn Drive, Rockville, Md. 20852 under ATCC No.: 67825. The cDNA, and fragments and derivatives thereof, can be used as a probe to isolate DNA sequences encoding for proteins similar to the receptor protein encoded by the cDNA. Namely, the cDNA of a human receptor corresponding to the mouse cDNA 4-1BB can be isolated from a human source using cDNA 4-1BB as a probe.
The receptor protein 4-1BB can be produced by: 1) inserting the cDNA of 4-1BB into an appropriate expression vector, 2) transfecting the expression vector into an appropriate transfection host, c) growing the transfected hosts in appropriate culture media and d) purifying the receptor protein from the culture media. The protein and fragments and derivatives can be used: 1) as a probe to isolate ligands to receptor protein 4-1BB, 2) to stimulate proliferation of B-cell""s expressing 4-1BB ligands, or 3) to block 4-1BB ligand binding. B-cells that have expressed a ligand to receptor protein 4-1BB are treated with cells that have expressed receptor protein 4-1BB and B-cell proliferation is induced. The use of 4-1BB to block 4-1BB ligand binding has practical application in the suppression of the immune system during organ transplantation. A similar costimulatory immune system pathway is being analyzed for this type of application. See xe2x80x9cMounting a Targeted Strike on Unwanted Immune Responsesxe2x80x9d, Jon Cohen, Science, Vol. 257, 8-7-92; xe2x80x9cLong Term Survival of Xenogeneic Pancreatic Islet Grafts Induced by CTLA4Igxe2x80x9d, Lenschow et al, Science Vol. 257, 7-8-92; and xe2x80x9cImmunosuppresion in Vivo by a Soluble Form of the CTLA-4 T Cell Activation Moleculexe2x80x9d, Linsley et al, Science Vol. 257 7-8-92.
A monoclonal antibody against 4-1BB was developed which specifically recognizes an epitope on the extracellular domain of receptor protein 4-1BB. The monoclonal antibody is produced from a hybridoma identified as 53A2 and deposited at the American Type Culture Collection at 12301 Parklawn Drive, Rockville, Md. 20852 under ATCC No.: HB-11248. The monoclonal antibody can be used to enhance T-cell proliferation by treating T-cells that have expressed receptor protein 4-1BB with antiCD3 monoclonal antibody.
Some tumors are potentially immunogenic but do not stimulate an effective anti-immune response in vivo. Tumors may be capable of delivering antigen-specific signals to T cells, but may not deliver the co-stimulatory signals necessary for full activation of T cells. Expression of the co-stimulatory ligand on B7 of melanoma cells was found to induce the rejection of a murine melanoma in vivo. (xe2x80x9cTumor Rejection After Direct Co-Stimulation of CD8+ T Cells by B7-Transfected Melanoma Cellsxe2x80x9d, Sarah E. Townsend and James P. Allison, Science Vol. 259, 1-5-93.). The monoclonal antibody of the present invention may be capable of the same effect as it is now known to enduce T cell proliferation and activation.
A fusion protein for detecting cell membrane ligands to receptor protein 4-1BB was developed. It comprises the extracellular portion of the receptor protein 4-1BB and a detection protein bound to the portion of the receptor protein 4-1BB. The portion of the receptor protein 4-1BB binds to the cell membrane ligands and binding can be detected by relative activity assays for the detection protein. The fusion protein is placed in the presence of a cell suspected to express the receptor protein 4-1BB. Then the cell is washed of any fusion protein not bound to the cell membrane ligands. Once the washed cells are placed in the presence of a substrate for the detection protein and the relative activity of the detection protein can be measured. The detection protein disclosed herein is alkaline phosphatase.
The primary object is to provide the teachings identifying the new receptor, 4-1BB as identified herein by its sequence.
Another object of the present invention is to provide teachings of how the new receptor may be used to isolate and identify corresponding molecules in related species.
Still another object of the teachings of the present invention is to teach the identification of the new receptor as reported herein.
Still another object of the teachings of the present invention is to teach the anti-4-1BB monoclonal antibody produced from the hybridoma 53A2 as reported herein.
Still another object of the teachings of the present invention is to teach a fusion protein comprising the extracellular portion of 4-1BB and a detection protein.
Still another object of the teachings of the present invention is to teach methods of using the cDNA 4-1BB, the receptor protein 4-1BB, the monoclonal antibody and the fusion protein.